Summary of Work: The goal of this project is to investigate the signaling pathways, particularly ion signaling pathways, that regulate apoptosis. We studied cells that responded differently to apoptotic induction following growth factor removal (i.e. low serum). Early stage preneoplastic, immortal cells (sup+), show high susceptibility to induction of apoptosis, whereas late stage preneoplastic cells (sup-) are relatively resistant to apoptosis following serum reduction to 0.2%. We are investigating whether altered Ca2+ homeostasis is causally involved in apoptosis. We have shown that a sustained increase in cytosolic free Ca2+ (Cai) does not precede apoptosis. Differences in endoplasmic reticulum (ER) calcium between sup+ cells and sup- were determined by measuring thapsigargin releasable Ca2+ in the presence of 10% and 0.2% serum. Sup+ cells had less thapsigargin releasable Ca2+ than sup- cells in 0.2% serum, consistent with the hypothesis that depletion of ER calcium plays a role in apoptosis. ER calcium depletion was observed consistently with induction of apoptosis, regardless of the agent or cell line used. Decreased calcium entry into the cells appears to be involved in the decreased ER calcium since raising extracellular Ca2+ to 3 mM blocked both the decrease in ER calcium and DNA fragmentation. Measurement of Ca2+ entry via Mn2+ uptake showed that in 0.2% serum the rate of thapsigargin induced Mn2+ entry in sup+ cells is approximately 50% less than that of sup- cells, suggesting that capacitative entry is reduced in sup+ cells in low serum and this could be an important factor in the observed decrease in ER calcium and apoptosis. Further supporting this concept, SKF 96365, inhibitor of capacitative calcium entry, induced apoptosis under conditions not normally causing apoptosis (e.g. SHE and sup- cells in 0.2% serum, and sup+ cells in 10% serum). We are investigating mechanism(s) that might be responsible for the altered calcium entry and apoptosis. We are also investigating mechanisms by which a decrease in ER calcium might cause apoptosis.